Ligand-activated peroxisome proliferator-activated receptor-δ and -γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

Abstract

Peroxisome proliferator-activated receptors (PPARs) inhibit lipopolysaccharide (LPS)-primed release of high mobility group box 1 (HMGB1), a late proinflammatory mediator, but the underlying molecular mechanism is not completely understood. In this study, we demonstrated that the inhibition of HMGB1 release by PPAR-δ and -γ is associated with the deacetylase activity of SIRT1. Ligand-activated PPAR-δ and -γ inhibited LPS-primed release of HMGB1, concomitant with elevation in SIRT1 expression and promoter activity. These effects were significantly reduced in the presence of small interfering (si)RNAs against PPAR, indicating that PPAR-δ and -γ are involved in both HMGB1 release and SIRT1 expression. In addition, modulation of SIRT1 expression and activity by siRNA or chemicals correspondingly influenced the effects of PPARs on HMGB1 release, suggesting a mechanism in which SIRT1 modulates HMGB1 release. Furthermore, we showed for the first time that HMGB1 acetylated in response to LPS or p300/CBP-associated factor (PCAF) is an effective substrate for SIRT1, and that deacetylation of HMGB1 is responsible for blockade of HMGB1 release in macrophages. Finally, acetylation of HMGB1 was elevated in mouse embryonic fibroblasts from SIRT1-knockout mice, whereas this increase was completely reversed by ectopic expression of SIRT1. These results indicate that PPAR-mediated upregulation of SIRT1 modulates the status of HMGB1 acetylation, which, in turn, has a critical role in the cellular response to inflammation through deacetylation-mediated regulation of HMGB1 release.

Figures

Figure 1

Ligand-activated PPARs inhibit LPS-induced release of HMGB1. (a) RAW 264.7 cells grown to 60% confluency were incubated in serum-free medium for 24 h, and then stimulated with LPS in the presence or absence of PPAR ligands for 24 h. (b and c) Cells were transfected with siRNA against PPAR-δ (b) or PPAR-γ (c), and grown for 38 h. After incubation in serum-free medium for 24 h, the cells were stimulated with LPS for 24 h in the presence or absence of GW501516 (b) or rosiglitazone (c). Equal volumes of conditioned media or aliquots of cell lysates were subjected to western blotting for determination of HMGB1 levels. An image analyzer was used to quantitate band intensity, and the ratios of HMGB1 to Ponceau S are shown. The results are expressed as means±S.E.M. (n=3). **P<0.01 compared with the untreated group; ##P<0.01 compared with the LPS-treated group; ††P<0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining or β-actin was used as a loading control

Figure 2

Acetylation is involved in the regulation of HMGB1 release. (a) RAW 264.7 cells incubated in serum-free medium for 24 h were pretreated with resveratrol or sirtinol for 1 h, and then stimulated with LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of HMGB1 released). (b) Cells transfected with SIRT1 siRNA or control siRNA were grown for 38 h, after which they were incubated in serum-free medium for 24 h, and then stimulated with LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of released HMGB1). Cell lysates were pulled down with anti-HMGB1 and immunoblotted with anti-acetyl-lysine to detect acetylated HMGB1. Each membrane was then stripped and re-probed for total HMGB1, as a loading control. For determination of released HMGB1, equal volumes of conditioned media were subjected to western blot analysis; Ponceau S staining was used as a loading control. Whole-cell lysates were subjected to Western blot analysis to determine the expression levels of HMGB1 (a) and SIRT1 (b). An image analyzer was used to quantitate band intensity, and the fold changes in the acetyl-HMGB1 to HMGB1 or HMGB1 to Ponceau S ratio are shown. The results are expressed as means±S.E.M. (n=3). **P<0.01, *P<0.05 compared with the untreated group; ##P<0.01, #P<0.05 compared with the LPS-treated group. Ponceau S staining or β-actin was used as a loading control

Figure 3

SIRT1 mediates the effects of PPAR-δ and -γ in the inhibition of LPS-primed release of HMGB1 through deacetylation. (a) RAW 264.7 cells grown to 60% confluency were incubated with serum-free medium for 24 h and then stimulated with LPS for the indicated times. (b) Cells incubated for 24 h in serum-free medium were stimulated with LPS for 6 h in the presence or absence of the ligand indicated. (c and d) Cells were transfected with SIRT1 siRNA and grown for 38 h, after which they were stimulated with LPS for 6 h in the presence or absence of GW501516 (c) or rosiglitazone (d). Whole-cell lysates were immunoprecipitated with anti-HMGB1, and then acetylated HMGB1 was detected by western blotting with an anti–acetyl-lysine antibody. An image analyzer was used to quantitate band intensity, and the ratios of acetylated HMGB1 to total HMGB1 are shown. The results are expressed as means±S.E.M. (n=3). *P<0.05 compared with the untreated group; #P<0.05 compared with the LPS-treated group; †P<0.05 compared with the group treated with LPS+GW501516 or rosiglitazone

Figure 4

Ligand-activated PPAR-δ and -γ upregulate SIRT1 expression. (a) RAW 264.7 cells were treated with GW501516 or rosiglitazone for the indicated times. Total RNA was extracted, and SIRT1 mRNA levels were determined by real-time PCR. (b) Cells were treated with GW501516 or rosiglitazone for the indicated times. (c and d) Cells transfected with siRNA against PPAR-δ (c) or PPAR-γ (d) were treated with the indicated PPAR ligand for 3 h. Aliquots of protein from cell lysates were analyzed by western blotting with anti-SIRT1 or anti-β-actin antibody. (e) Cells pretreated with cycloheximide or actinomycin D for 30 min were incubated with 100 nM GW501516 or 10 μM rosiglitazone for 1 h. Total RNA was extracted and reverse transcribed into cDNA. Equal amounts of cDNA were diluted and amplified by real-time PCR. The fold change in SIRT1 cDNA relative to the GAPDH control was determined and plotted. (f) Cells transfected with a SIRT1 promoter-reporter construct and pSV β-gal were grown for 38 h and then exposed to the indicated ligand for 1 h. The luciferase activity was normalized to β-galactosidase activity, and the data are expressed as means±S.E.M. (n=5). *P<0.05 compared with the untreated group; #P<0.05 compared with the GW501516- or rosiglitazone-treated group

Figure 5

SIRT1 is essential for the inhibition of LPS-induced HMGB1 release by PPAR-δ and -γ. (a and b) RAW 264.7 cells pretreated with resveratrol (a) or sirtinol (b) for 1 h were stimulated with LPS in the presence or absence of GW501516. (c and d) Cells pretreated with resveratrol (c) or sirtinol (d) for 1 h were incubated with LPS in the presence or absence of rosiglitazone. After incubation for 24 h, equal volumes of conditioned media were analyzed by western blotting with an anti-HMGB1 antibody. An image analyzer was used to quantitate band intensity, and the ratios of HMGB1 to Ponceau S are shown. The results are expressed as means±S.E.M. (n=3). **P<0.01 compared with the untreated group; ##P<0.01 compared with the LPS-treated group; ††P<0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining was used as a loading control

Figure 6

Downregulation of SIRT1 abrogates the prevention of PPAR-δ and -γ against HMGB1 release. (a) RAW 264.7 cells grown to 60% confluency were incubated with serum-free medium for 24 h, and then stimulated with LPS in the presence or absence of the indicated ligand for 24 h. Aliquots of protein from cell lysates were analyzed by western blotting with anti-SIRT1 or anti-β-actin antibody. (b and c) Cells transfected with siRNA against SIRT1 were stimulated with LPS in the presence or absence of the GW501516 (b) or rosiglitazone (c) for 24 h. Equal volumes of conditioned media were analyzed by western blotting with anti-HMGB1 antibody. An image analyzer was used to quantitate band intensity, and the fold changes in the HMGB1 to Ponceau S or SIRT1 to β-actin ratio are shown. The results are expressed as means±S.E.M. (n=3). **P<0.01 compared with the untreated group; ##P<0.01, #P<0.05 compared with the LPS-treated group; ††P<0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining or β-actin was used as a loading control

Figure 7

SIRT1-mediated deacetylation of HMGB1 is a critical factor in the PPAR-δ/γ–mediated inhibition of HMGB1 release. (a) RAW 264.7 cells were transfected with empty vector (pcDNA3.1/Myc) or pcDNA3.1-Myc-SIRT1. (b) Cells were transfected with empty vector (pcDNA3.1/HA) or pcDNA3.1-HA-PCAF. (c) Cells were transfected with empty vector (pcDNA3.1), pcDNA3.1-Myc-SIRT1, or pcDNA3.1-HA-PCAF. After incubation for 38 h, cells were maintained in serum-free medium for 24 h, and then stimulated with or without LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of released HMGB1). Cell lysates were pulled down with anti-HMGB1 and immunoblotted with anti-acetyl-lysine to detect acetylated HMGB1. Each membrane was then stripped and re-probed for HMGB1, as a loading control. For determination of released HMGB1, equal volumes of conditioned media were subjected to Western blot analysis; Ponceau S staining was used as a loading control. Whole-cell lysates were subjected to western blot analysis with an anti-HMGB1, anti-SIRT1, anti-Myc, or anti-HA antibody, as appropriate, to determine the expression levels of HMGB1 and SIRT1 (a and c) or PCAF (b and c)

Figure 8

Acetylated HMGB1 is an effective substrate for SIRT1. (a) HEK293T cells were transfected with empty vector (pcDNA3.1), pcDNA3.1-Flag-HMGB1, or pcDNA3.1-Myc-SIRT1. (b) HEK293T cells were transfected with pcDNA3.1-HA-PCAF or increasing amounts (2 μg and 4 μg) of pcDNA3.1-Myc-SIRT1. After incubation for 48 h, the cells were stimulated with (a) or without (b) LPS for 3 h. (c) Whole-cell lysates from SIRT1-knockout (SIRT1−/−) or wild-type (SIRT1+/+) MEFs were pulled down with anti-HMGB1 and analyzed by western blotting with anti-acetyl-lysine, anti-HMGB1, or anti-SIRT1 antibody to detect acetylated HMGB1 or total HMGB1 and SIRT1. (d) SIRT1−/− MEFs were transfected with empty vector (pcDNA3.1-Myc) or pcDNA3.1-Myc-SIRT1 and incubated for 48 h, after which the cells were harvested and subjected to immunoprecipitation with anti-HMGB1 antibody. Acetylated HMGB1 or total HMGB1 and SIRT1 were detected by western blot analysis. β-actin was used as a loading control